Isolation of NEDDylated Proteins in Human Cells

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 832)

Abstract

Identification of substrates for post-translational modification with ubiquitin and ubiquitin-like molecules is a key in our understanding of the role of this modification in the regulation of biological processes. The ubiquitin-like molecule NEDD8 plays a vital role in cell growth, viability, and development. In this ­chapter, we describe a method for efficient isolation and detection of NEDDylated proteins in human cells.

Key words

NEDD8 Histidine Nickel purification Human cell lines Western blotting 
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Notes

Acknowledgements

Research in DPX laboratory is supported by the Association for International Cancer Research (AICR) and INSERM. D.P.X. is an AICR Research Fellow.

References

  1. 1.
    Kamitani T, Kito K, Nguyen HP, Yeh ET (1997) Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein. J Biol Chem 272:28557–28562.PubMedCrossRefGoogle Scholar
  2. 2.
    Pozo JC, Timpte C, Tan S et al (1998) The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science 280:1760–1763.PubMedCrossRefGoogle Scholar
  3. 3.
    Osaka F, Saeki M, Katayama S et al (2000) Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast. Embo J 19:3475–3484.PubMedCrossRefGoogle Scholar
  4. 4.
    Ou CY, Lin YF, Chen YJ, Chien CT (2002) Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development. Genes Dev 16:2403–2414.PubMedCrossRefGoogle Scholar
  5. 5.
    Jones D, Candido EP (2000) The NED-8 conjugating system in Caenorhabditis elegans is required for embryogenesis and terminal differentiation of the hypodermis. Dev Biol 226:152–165.PubMedCrossRefGoogle Scholar
  6. 6.
    Tateishi K, Omata M, Tanaka K, Chiba T (2001) The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice. J Cell Biol 155:571–579.PubMedCrossRefGoogle Scholar
  7. 7.
    Kerscher O, Felberbaum R, Hochstrasser M (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol 22:159–180.PubMedCrossRefGoogle Scholar
  8. 8.
    Xirodimas DP (2008) Novel substrates and functions for the ubiquitin-like molecule NEDD8. Biochem Soc Trans 36:802–806.PubMedCrossRefGoogle Scholar
  9. 9.
    Rabut G, Peter M (2008) Function and regulation of protein neddylation. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep 9:969–976.PubMedCrossRefGoogle Scholar
  10. 10.
    Huang DT, Ayrault O, Hunt HW et al (2009) E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification. Mol Cell 33:483–495.PubMedCrossRefGoogle Scholar
  11. 11.
    Lyapina S, Cope G, Shevchenko A et al (2001) Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science 292:1382–1385.PubMedCrossRefGoogle Scholar
  12. 12.
    Schwechheimer C, Serino G, Callis J et al (2001) Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response. Science 292:1379–1382.PubMedCrossRefGoogle Scholar
  13. 13.
    Liakopoulos D, Doenges G, Matuschewski K, Jentsch S (1998) A novel protein modification pathway related to the ubiquitin system. Embo J 17:2208–2214.PubMedCrossRefGoogle Scholar
  14. 14.
    Pan ZQ, Kentsis A, Dias DC et al (2004) Nedd8 on cullin: building an expressway to protein destruction. Oncogene 23:1985–1997.PubMedCrossRefGoogle Scholar
  15. 15.
    Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6:9–20.PubMedCrossRefGoogle Scholar
  16. 16.
    Skaar JR, Florens L, Tsutsumi T et al (2007) PARC and CUL7 form atypical cullin RING ligase complexes. Cancer Res 67:2006–2014.PubMedCrossRefGoogle Scholar
  17. 17.
    Soucy TA, Smith PG, Milhollen MA et al (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458:732–736.PubMedCrossRefGoogle Scholar
  18. 18.
    Liu G, Xirodimas DP (2010) NUB1 promotes cytoplasmic localization of p53 through cooperation of the NEDD8 and ubiquitin pathways. Oncogene 29:2252–2261.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Wellcome Trust Centre for Gene Regulation and Expression, College of Life SciencesUniversity of DundeeDundeeUK
  2. 2.Centre de Recherche de Biochimie Macromoléculaire – UMR 5237, CNRSMontpellierFrance

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